I came across a paper where a gene is being amplified by many pairs of primers. After amplification, all the PCR products were sequenced and were assembled. I wonder what was the reason behind using many sets of primers to amplify and then assembling them and why did not the single set of primer pairs were used for amplification?
Maybe the gene was too large to be efficiently amplified in one step, particularly if it contained regions that are hard to copy byDNA polymerase (palindromes, high GC...) ?
How old is the paper? It used to be hard to amplify PCR products above a certain size. I've been doing this long enough that 2kb was considered "large" when I started.
Now, it's usually very easy to amplify 10kb or larger products with a long-read polymerase. But not everyone knows that's possible and sometimes it simply doesn't work.
Was this a key part of the paper or just commentary on their methods?
We did something similar for sequencing a gene. We used two pairs of primers, one to amplify the gene + 5' region, one for gene + 3'.
You don't know how the quality of the sequencing will be, so we did this to be more sure. We amplified the gene twice, sequenced, and assembled.
Pierre Béguin thank you for the response. The original length of the gene was 3465 bp, but the amplified and assembled gene was 1469 bp. Further, the gene had 34% of GC content and there are 11 palindromic sequence of 4 bp, 2 palindromic sequences of 5 bp, 1 palindromic sequence of 6 bp and 1 palindromic sequence of 7 bp.
Katie A Burnette thank you for the response. This paper was published in 2012. the gene was 3465 bp. But the amplified and assembled sequence was 1469 bp. It is one of the preliminary step for further experiment.
Hey,
It might have several reasons.
But mainly, before there were Q5 like polymerases, polymerases that we used were not that efficient to amplify that long sequences.
Or if I understand correct from essambling, if there is a cloning procedure and if the gene contains an internal restriction site which is being used in restriction cloning. You can seperate from the restriction site by PCR, and you can clone 5`and 3" of the gene by restriction-ligation reaction. And the internal site by blunt end cloning.
Yagiz
Likhith R K < the gene was 3465 bp. But the amplified and assembled sequence was 1469 bp. >
The 3465 bp gene might contain both exons and introns. The 1465 bp gene only has the exons? But, I don't know why they did it this way, instead of using cDNA, if it is the case. You need to know their research goal first. Could you attach the paper here?
Hello Yuan-Yeu Yau, this is the DOI of the article I was talking about.
10.1007/s11103-012-9946-6
Likhith R K I read the paper (2012) DOI: 10.1007/s11103-012-9946-6.
Yes, as I pointed out earlier, the authors were trying to figure out a cDNA and its full-length genomic DNA. This is what they do:
1. They had a cDNA fragment (sequenced) (called 'X') from their cDNA library.
2. They blasted X and matched to part of a full-length cDNA (unknown, in a database, called "Y")
3. Since cDNA Y is full length, they designed primers (several pairs) from Y. Then used these primer pairs to amplify genomic DNA. They obtained several PCR products (the PCR products contain exons or/and introns).
4. They then *assembled* DNA sequences together in silico (by computer), to figure out their gene-of-interest full length genomic DNA. (NOT assemble the physical PCR fragments)
5. Paper attached.
Yuan-Yeu Yau If I want to amplify this gene, can you please tell me what am I supposed to do?
Likhith R K
Design primers to cover the start codon and stop codon, and do a PCR amplification using a hi-fi Taq DNA polymerase. I think the DNA sequence (database access number) is listed in the paper you attached. Do you want to amplify the full-length genomic DNA of the gene or its cDNA?
Yuan-Yeu Yau the gene is over-expressed only upon virus inoculation. So, I think I should be amplifying the cDNA. What do you think of it? Any suggestions regarding this??
Likhith R K Do you want to overexpress this gene in a plant? You can use cDNA. cDNA is shorter (~1.5 kb) and easier to be amplified and cloned, compared to use genomic DNA (~3.5 kb). To get the cDNA, isolate total RNA and do a RT-PCR. Then cloned the cDNA into a binary vector for transformation. Which plant species you want to overexpress this gene--dicot? monocot? tomato?
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